As an unexpected result of our study of effects of the somatomedins on muscle cell differentiation, we have recently discovered a novel biological agent (secreted by the Buffalo Rat Liver cell line in serum-free medium) which exhibits potent and reversible inhibition of muscle cell differentiation in culture. The material is trypsinsensitive, non-dialyzable, and relatively heat- and acid-stable. We have achieved a substantial (750-fold) purification and some characterization of this protein, which we designate Differentiation Inhibitor. At concentrations at which it exhibits no detectable lectin or mitogen activity, it inhibits all tested aspects of differentiation in all myoblast preparations (L6 line, rat, chick, and Japanese quail) we have used. We now propose to complete purification of the Differentiation Inhibitor and investigate its nature and actions. We will determine whether or not it corresponds to the differentiation-inhibiting component of fetal bovine serum and/or chick embryo extract, and investigate its role in fetal development and satellite cell formation. We will establish the time course of its actions, measure its interactions with myoblasts, and attempt to determine its biochemical mechanism of action. We will also use it as a tool to study control of muscle cell differentiation. For this, we have a system which is uniquely well-defined: in serum-free medium, we can stimulate (with MSA, a somatomedin also produced by BRL cells) or block (with Differentiation Inhibitor) differentiation of cloned L6 myoblasts. We will also continue our studies on the actions of the somatomedins in muscle, in general shifting emphasis from tissue culture to effects on growth and development of whole animals. We will use antibodies to MSA to investigate its role in fetal development, and will use the large quantities of MSA obtained as a byproduct of Differentiation Inhibnitor production to investigate its effect on growth of hypophysectomized rats.